Interaction of Enterohemorrhagic Escherichia coli (EHEC) with Mammalian Cells: Cell Adhesion, Type III Secretion, and Actin Pedestal Formation

Abstract

Infection by the food‐borne pathogen enterohemorragic Escherichia coli (EHEC) can lead to diarrhea, hemorrhagic colitis, and, in the most serious cases, renal failure. A critical step in colonization is the unusual interaction between EHEC and the intestinal epithelium. EHEC is able to adhere to mammalian cells, and then, by injecting bacterial proteins, or “effectors,” into the host cell via a type III secretion system, induces the formation of attaching and effacing (AE) lesions characterized by the accumulation of filamentous actin directly beneath bound bacteria. This unit describes methods to evaluate the ability of EHEC to adhere to cultured mammalian cells, to secrete type III effectors, and to promote the formation of actin “pedestals.” These methods can not only be used to evaluate the contribution of specific EHEC gene products to adherence, type III secretion, and mammalian cell signaling, but also facilitate the analysis of the eukaryotic pathways controlling fundamental cellular processes such as actin assembly.

The Bacteroidetes Q-Rule: Pyroglutamate in Signal Peptidase I Substrates

“…A downstream 2835 bp fragment, comprising the 5′ sequence of the RgpA gene together with 388 bp of its proposed promotor was amplified with primers RgpA_Dw_F and RgpA_Dw_R. The tetracycline ( tetQ ) resistance cassette was amplified from the pT-COW plasmid ( Belanger et al, 2007 ) with primers Tet_BamHI_F and Tet_SalI_R. The backbone, the pUC19 plasmid, was linearized by PCR reaction with primers puc_EcoRI_R and puc_HindIII_F.…”

Shiga Toxins and the Pathophysiology of Hemolytic Uremic Syndrome in Humans and Animals

“…Several recent reviews discuss the molecular basis for bacterial success and toxin biochemistry [6,7,8]. Bacterial colonization of the intestine with attaching and effacing lesions support type III secretion of toxins into the vasculature [6]. Micropinocytosis may contribute as toxin antigen is observed in patient gut epithelial cells in the absence of A/E lesions [9].…”